Fatty acid binding protein 1 and fatty acid synthetase over-expression have differential effects on collagen III synthesis and cross-linking in Zongdihua pig primary adipocytes

The purpose of this study was to determine whether FABP1 and FAS regulate expression of collagen and its crosslinking via lysyl oxidase in isolated adipocytes from Zongdihua pigs. We aimed to identify biochemical processes affecting meat quality using molecular tools to provide a basis for breeding improvement of these animals. We measured expression levels of FABP1 and related genes using qRT-PCR in longissimus dorsi muscle and subcutaneous adipose tissues. Primary adipocytes from fat tissues were isolated and FABP1 and FAS were over-expressed from recombinant plasmids. Sequence analysis of the cloned genes indicated that FABP1 encodes a hydrophobic protein of 128 amino acids and contained 12 predicted phosphorylation sites and no transmembrane regions. The basal levels of FABP1 and FAS expression in pig tissues were 3–3.5-fold higher in subcutaneous fat compared with muscle (P < 0.01). Recombinant expression plasmids were successfully transfected into the cloned preadipocytes and (a) over-expression of FAS resulted in significantly increased expression of COL3A1 (P < 0.05) and significantly inhibited lysyl oxidase LOX expression (P < 0.01); (b) over-expression of FABP1 significantly increased COL3A1 expression (P < 0.01) and significantly inhibited LOX expression (P< 0.05) and significantly reduced lysyl oxidase activity (P < 0.01). Therefore, FAS enhanced FABP1 expression resulting in increased collagen accumulation and this preliminarily suggested that FAS and FABP1 can serve as fat-related candidate genes and provide a theoretical basis for the study of fat deposition in Zongdihua pigs.

enhancing the expression of FAS and FABP1 increases collagen accumulation and this preliminarily suggests that FAS and FABP1 can serve as fat-related candidate genes providing a theoretical basis for the study of fat deposition in Zongdihua pigs. The manuscript brings interesting results, however, to be accepted for publication, it needs major changes and corrections. Below are suggestions to make the manuscript suitable for publication and more informative to the reader.

General suggestions
-Write all gene names in italics. Example: FABP1 -Please review the text for language.
-Some words are used in different ways in the manuscript such as crosslinking/crosslinking, Over-expression/Overexpression…Correct this, please.
-The introduction needs to be rewritten, addressing the issues related to the manuscript more deeply. In addition, give a clear hypothesis and objective in the introduction section.
-Discussion needs to be rewritten, addressing more deeply the observed results.

Abstract
-Line 26 -Remove the with in the sentence "FAS encodes 333 amino acid hydrophobic protein containing with 26 phosphorylation sites and 0 transmembrane regions".
-Line 27 -Change the following sentence "The basal levels of FABP1 and FAS in pig tissues expression were 3 -3.5-fold higher in subcutaneous fat compared with muscle (P < 0.01)" to "The basal levels of FABP1 and FAS expression in pig tissues were 3 -3.5fold higher in subcutaneous fat compared with muscle (P < 0.01)".
-Line 33-I believe you forgot to add "FAS" in this sentence "Therefore, enhancing FABP1 expression increases collagen accumulation...". Am I correct? Because the overexpression of FAS and FABP1 resulted in a significantly increased expression of the COL3A1 gene.
-Keywords must be arranged in alphabetical order. It is not recommended to add words that are already in the title of the manuscript such as Zongdihua pig.

Introduction
-Add the title "Introduction" before the text in the line 40.
-You could improve your introduction by talking more about the breed (Zongdihua pig), mainly about the meat traits. It could also present some preliminary studies that address the evaluated genes deeply. All these issues need to be better integrated. In addition, the objective of the work should be better presented in this introduction.

Materials and methods
-Line 58 -Correct to Ethics Statements.
-Why is the topic "Ethics Statements" highlighted in the text? -Please, I ask you to correct the text in relation to the language used. Some sentences are poorly worded. -Is the Y-axis legend correct in Figure 9?

Discussion
-Line 225:226 -Change the sentence "In this study, we found that FABP1 and FAS gene expression were significantly higher than for the control muscle tissue" to "In this study, we found that FABP1 and FAS gene expression were significantly higher in subcutaneous fat than for the control muscle tissue" -Line 232 -Alter LOC gene to LOX.
-Your discussion of the results is superficial, you need to go deeper into the effect of the FAS and FABP1 genes on the expression of COL3A1 and LOX and the production of collagen. In addition to the effects of these genes on the deposition of fat and on meat traits.
-Line 231:233-"Our results agreed with previous studies indicating that COL3A1 and LOC gene expression in subcutaneous fat are significantly higher and lower than in longissimus dorsi muscle tissues, respectively." - Figure 1 shows greater expression of both the COL3A1 and LOX genes in subcutaneous fat in relation to Longissimus dorsi.
-You have not discussed deeply the effects of the expression of these genes on meat quality in the breed studied.